Fluorinated compounds have numerous applications in medicine as therapeutic and diagnostic agents. Fluorine has a van der Waals radius (1.2 Å) similar to hydrogen (1.35 Å), and hydrogen replacement with fluorine typically does not cause significant conformational changes. Fluorinated compounds are often biologically inert. Furthermore, the carbon-fluorine bond strength (460 kJ/mol in CH3F) exceeds that of equivalent C—H bonds.
The high sensitivity of 19F to surrounding environments, 100% natural abundance and high sensitivity to NMR detection (83% that of 1H) has made 19F NMR spectroscopy useful for investigating protein structure and dynamics. Gerig, Fluorine NMR of Proteins, Progress in Nuclear Magnetic Resonance Spectroscopy 26(4), 293-370 (1994). The simplicity of observing hypersensitive 19F chemical shifts by NMR makes it an exquisite tool for monitoring protein movements resulting from small molecule binding, covalent modification, or protein interactions. Bourret, et al., Activation of The Phosphosignaling Protein CheY. II. Analysis of Activated Mutants by 19F NMR and Protein Engineering, J. Biol. Chem. 268(18), 13089-96 (1993); Luck, et al., 19F NMR Studies of The D-Galactose Chemosensory Receptor. 1. Sugar Binding Yields a Global Structural Change, Biochemistry 30(17), 4248-56 (1991); Hinds, et al., 19F NMR Studies of Conformational Changes Accompanying Cyclic AMP Binding to 3-Fluorophenylalanine-Containing Cyclic AMP Receptor Protein from Escherichia coli, Biochem. J. 287 (Pt. 2), 627-32 (1992); Luck, et al., 19F NMR Studies of The Recombinant Human Transferrin N-Lobe and Three Single Point Mutants, Magn. Reson. Chem. 35, 477-81 (1997). The ability to uniformly label any single site in a protein in vivo will enable the study of large proteins with unprecedented chemical clarity.
Because 19F-labeled proteins have also seen interest in solid state membrane protein studies, folding studies, protein stabilization and probing disease states with 19F MRI, a general method for genetically incorporating a 19F-label into proteins of any size in Escherichia coli would have broad application. See, e.g., Bai, et al., Side Chain Accessibility and Dynamics in The Molten Globule State of alpha-Lactalbumin: A 19F-NMR Study, Biochemistry 39(2), 372-80 (2000); Vaughan, et al., Difluoromethionine as A Novel 19F NMR Structural Probe for Internal Amino Acid Packing in Proteins, J. Am. Chem. Soc. 121(37), 8475-78 (1999); Higuchi, et al., 19F and 1H MRI Detection of Amyloid beta Plaques in Vivo, Nat. Neurosci. 8(4), 527-33 (2005); Bann, et al., Folding and Domain-Domain Interactions of The Chaperone PapD Measured by 19F NMR, Biochemistry 43(43), 13775-86 (2004); Hoeltzli, et al., Refolding of [6-19F]Tryptophan-Labeled Escherichia coli Dihydrofolate Reductase in The Presence of Ligand: A Stopped-Flow NMR Spectroscopy Study, Biochemistry 37(1), 387-98 (1998). Although 19F NMR is a powerful technique for monitoring protein conformational changes and interactions, the inability to site-specifically introduce fluorine labels into proteins of interest severely limits its applicability. Ulrich, Solid State 19F NMR Methods for Studying Biomembranes, Progress in Nuclear Magnetic Resonance Spectroscopy 46, 1-21 (2003).
Drawbacks of current methods for the incorporation of fluorinated amino acids into proteins are numerous. Semisynthetic incorporation enables high fidelity at specific sites but becomes impractical when medium to large proteins are needed. The use of natural translational machinery to force fluorinated mimics of Tyr, Trp, Phe, Met, and Leu into their natural codons may produce large proteins, but altering all locations of one amino acid simultaneously in large proteins results in structural perturbation and overlapping of 19F signals. Danielson, et al., Use of 19F NMR to Probe Protein Structure and Conformational Changes, Annu. Rev. Biophys. Biomol. Struct. 25, 163-95 (1996); Vaughan, et al., Difluoromethionine as A Novel 19F NMR Structural Probe for Internal Amino Acid Packing in Proteins, J. Am. Chem. Soc. 121(37), 8475-78 (1999); Feeney, et al., 19F Nuclear Magnetic Resonance Chemical Shifts of Fluorine Containing Aliphatic Amino Acids in Proteins: Studies on Lactobacillus casei Dihydrofolate Reductase Containing (2S,4S)-5-Fluoroleucine, J. Am. Chem. Soc. 118(36), 8700-06 (1996); Duewel, et al., Incorporation of Trifluoromethionine into A Phage Lysozyme: Implications and A New Marker for Use in Protein 19F NMR, Biochemistry, 36(11), 3404-16 (1997). Relying on natural machinery also means that incorporation of fluorinated mimics rarely approaches 90% and they are incorporated at different levels throughout the protein due to variation in codon usage.
A need exists for a reliable method for site-specific incorporation of fluorinated amino acids into proteins that does not suffer from the deficiencies of the prior art.